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Biochem. J. (2009) 418, 39­47 (Printed in Great Britain) doi:10.1042/BJ20081256

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Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family
Elien VANDERMARLIERE*, Tine M. BOURGOIS, Martyn D. WINN, Steven VAN CAMPENHOUT, Guido VOLCKAERT, Jan A. DELCOUR§, Sergei V. STRELKOV*, Anja RABIJNS* and Christophe M. COURTIN§1
*Laboratory for Biocrystallography, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Herestraat 49, O&N II, bus 822, 3000 Leuven, Belgium, Laboratory of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, bus 2462, 3001 Leuven, Belgium, Computational Science and Engineering Department, STFC Daresbury Laboratory, Daresbury, Warrington WA4 4AD, U.K., and §Laboratory of Food Chemistry and Biochemistry, Department of Microbial and Molecular Systems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, bus 2463, 3001 Leuven, Belgium

AXHs (arabinoxylan arabinofuranohydrolases) are -L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a twodomain structure for this enzyme: a catalytic domain displaying a five-bladed -propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a -sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic

stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, -xylosidase and -L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone. Key words: arabinoxylan arabinofuranohydrolase, Bacillus subtilis, crystallography, enzyme­substrate complex, family 43 glycoside hydrolase, substrate-binding mechanism.

INTRODUCTION

The use of renewable sources as an alternative to petroleumderived fuels and materials is receiving more and more research attention. At the moment, the most common renewable fuel is ethanol derived from cereal starch. Because of limitations in cereal supply, a better utilization of the whole kernel is being explored, with a focus on hemicellulose [1,2]. An important hemicellulose is arabinoxylan, which is composed of a linear backbone of -1,4-linked D-xylopyranosyl units that can be O-2 and/or O-3 substituted with L-arabinofuranosyl units. Enzymatic degradation of arabinoxylan into its building blocks requires a set of xylanolytic enzymes. Endoxylanases and -xylosidases hydrolyse the xylan backbone and act in synergy with arabinofuranosidases, which remove the arabinofuranosyl units [3]. These enzymes are thus important players in the conversion of heteroxylan into xylose and arabinose, which can then be used either for the production of ethanol or as platform molecules. -L-Arabinofuranosidases (EC 3.2.1.55) are classified according to their substrate specificity. Type A arabinofuranosidases are only active against small substrates such as 4-nitrophenyl -L-arabinofuranoside and short-chain AXOSs (arabinoxylooligosaccharides). Type B arabinofuranosidases can also hydrolyse polymeric substrates such as branched arabinan and arabinoxylan [4]. Some type B arabinofuranosidases specifically cleave arabinofuranosyl units from arabinoxylan and hence are termed AXH (arabinoxylan arabinofuranohydrolase) [5]. The latter type can be further divided into two and possibly three groups. AXH-m releases arabinose only from mono-substi-

tuted xylose units, whereas AXH-d releases it only from double´ substituted xylose units [6,7]. Ferre et al. [8] suggested that AXH from barley malt releases arabinose from both single- and doublesubstituted xylose units and therefore can be classified as AXHmd. Based on sequence similarity, the currently known arabinofuranosidases have been classified into five GH (glycoside hydrolase) families. Those found in GH families 3, 51 and 54 hydrolyse the glycosidic bond with retention of the anomeric configuration. Those in GH families 43 and 62 invert the anomeric configuration on hydrolysis [9,10]. To date, the only known structures of arabinofuranosidases are those of arabinofuranosidases belonging to GH families 51 and 54, displaying a ( / )8 -barrel and a -sandwich respectively [11,12]. Recently, Bourgois et al. [13] have characterized XynD from Bacillus subtilis subspecies subtilis A.T.C.C. 6051, which was previously predicted to be a member of GH family 43 displaying endoxylanase activity supplemented with arabinofuranosidase co-activity. Careful biochemical analysis characterized it as an arabinoxylan arabinofuranohydrolase that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues, i.e. as an AXH-m2,3 [13]. Within GH family 43, structures of xylosidases from Geobacillus stearothermophilus [14], Bacillus halodurans, B. subtilis and Clostridium acetobutylicum are available. Also -L-arabinanases from Cellvibrio japonicus [15] and Bacillus thermodenitrificans [16] and a bifunctional xylosidase/ -L-arabinofuranosidase from Selenomonas ruminantium [17] belonging to GH family 43 had their structure determined. Together with GH families 32, 62 and 68, their catalytic

Abbreviations used: AXH, arabinoxylan arabinofuranohydrolase; AXOS, arabinoxylo-oligosaccharide; Bs AXH-m2,3, Bacillus subtilis AXH-m2,3; CBM, carbohydrate-binding module; GH, glycoside hydrolase. 1 To whom correspondence should be addressed (email Christophe.Courtin@biw.kuleuven.be).
c The Authors Journal compilation c 2009 Biochemical Society

Biochemical Journal


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E. Vandermarliere and others

domain shares a five-bladed -propeller fold which was first seen for tachylectin [18]. Unlike the classical organization of the active site of inverting enzymes, which consists of a proton donor (the general acid) and a nucleophile (the general base), a third carboxylate was found to be essential for activity in GH family 43 enzymes. This residue is responsible for the pK a modulation of the general acid and correct orientation of both the proton donor and substrate [14]. Although the resolved -Larabinanase structures show no associated CBM (carbohydratebinding module), other GH family 43 members contain a CBM. For BsAXH-m2,3 (B. subtilis AXH-m2,3), it has been proposed, based on sequence similarity, that this CBM belongs to CBM family 6 [13]. Members of this family display a -sandwich fold: a lectin-like -jelly roll consisting of five antiparallel -strands packed against four antiparallel -strands. Like other CBMs, their function is to increase the effective concentration of the active site on polymeric substrate [19,20]. Since -xylosidases, -L-arabinanases and bifunctional xylosidases/ -L-arabinofuranosidases also belong to GH family 43, they not only have their structure in common, but also an analogous reaction mechanism as their catalytic residues occur at an analogous position [10]. However, unlike -xylosidases and -L-arabinanases, which are exo-acting enzymes, AXHm2,3 releases the arabinose substituents of arabinoxylan, so we can expect differences in the substrate-binding mechanism. To understand the binding of substrate, knowledge of the threedimensional structure is essential. For -xylosidase and -Larabinanase, the structure in complex with xylobiose [14] and the structure in complex with arabinohexaose [15] respectively are available and will be used here as reference structures. No structure of an AXH-m2,3 is available. We have previously reported the crystallization and preliminary X-ray analysis of BsAXH-m2,3 in complex with xylotriose [21]. Here, we describe the crystal structure of BsAXH-m2,3, the first structure of a GH family 43 arabinofuranosidase, and the first structure of an AXH overall, in complex with several substrates. The structure allows in-depth comparison of the substrate-binding mechanism between the GH family 43 classes.
EXPERIMENTAL Crystallization and data collection

Data from BsAXH-m2,3 crystals soaked in xylotriose and xylotetraose were collected on beamline BW7a of DESY (EMBLHamburg, Hamburg, Germany) at cryogenic temperature and processed using the HKL suite of programs [22]. Datasets of native BsAXH-m2,3 crystals and those soaked in cellotetraose and AXOS-4-0.5 were collected on beamline X10SA of SLS (Swiss Light Source; Villigen, Switzerland). These data were processed with iMosflm [23,24] and scaled and merged with Scala [25]. For all structures, 5 % of the observations were set aside for cross-validation analysis. These sets are all equal to the set of observations of the data collected of the BsAXH-m2,3 crystal soaked in xylotriose.
Structure solution and refinement

Recombinant AXH-m2,3 from B. subtilis was expressed and purified as described by Bourgois et al. [13]. Crystallization and data collection of BsAXH-m2,3 crystals soaked in xylotriose were reported earlier [21]. In short, thick needle-like crystals were grown in 4.0 M sodium formate. Refinement of this condition using the Additive screen (Hampton Research) resulted in several conditions in which rod-like crystals could be grown (Table 1). All these different crystals belong to the same space group and were used at random to prepare soaks with several sugars. Prior to data collection, the crystals were transferred briefly into cryoprotectant composed of 4.0 M sodium formate supplemented with 30 % (v/v) glycerol and a saturated concentration of xylotriose (Megazyme, Bray, Ireland) [21]. Analogous soaking experiments were performed using xylotetraose (Megazyme), cellotetraose (Sigma­Aldrich, St. Louis, MO, U.S.A.) and a mixture of AXOSs with an average degree of polymerization of 4 and an arabinose-to-xylose ratio of 0.5 (AXOS-4-0.5). These AXOSs were derived from WPC (wheat pentosan concentrate; Pfeifer & Langen, Dormagen, Germany) and were kindly made available by Katrien Swennen (Laboratory of Food Chemistry and Biochemistry, K.U. Leuven). In all cases, a saturated solution was used and soaking times ranged from 1 to 5 min.
c The Authors Journal compilation c 2009 Biochemical Society

All further computing was done using the CCP4 suite [26] unless stated otherwise. Due to the relatively low sequence similarity {25 % when comparing the catalytic domain with that of Ce. japonicus -L-arabinanase (PDB entry 1gyh) [15] and 35 % when comparing the CBM with B. halodurans CBM family 6 (PDB entry 1w9t) [27]}, phasing the structure of BsAXHm2,3 by molecular replacement presented a challenging problem. Eventually, the structure of BsAXH-m2,3 soaked in xylotriose was solved with an automatic approach: MrBUMP [28] implemented in the CCP4 interface, and using Phaser [29] as the underlying molecular replacement program, was used to position models of the two domains. An initial attempt at automated model rebuilding with ARP/wARP [30] using the sequence of BsAXH-m2,3 failed. Phase improvement of the molecular replacement solution was then carried out using artificial phase extension and dynamic density modification as implemented in the latest version of ACORN [31]. This step improved the phases sufficiently for ARP/wARP [30] to build and dock 93 % of the residues. The co-ordinates were evaluated using Coot [32] and few corrections were made manually. The refined structure was then used to solve the structure of the native protein and the remaining substrate complexes using Phaser. After several cycles of refinement, residual positive density in the F o ­ F c and 2F o ­ F c electron density revealed the presence of several bound sugar molecules. These were inserted manually followed by further refinement and model building using Refmac5 [33] and Coot respectively. The final structures were evaluated using Molprobity [34]. All data collection and refinement statistics are shown in Table 1. Figures 1­5 were drawn using the program PyMOL (DeLane Scientific; http://pymol.sourceforge.net/).
RESULTS AND DISCUSSION The overall structure

The structure of AXH-m2,3 from B. subtilis was determined by molecular replacement using the Ce. japonicus -L-arabinanase (PDB entry 1gyh) [15] and B. halodurans CBM family 6 (PDB entry 1w9t) [27] structures as search models for the catalytic domain and CBM respectively. Several soaking experiments were performed to gain insight into the binding of substrate. All structures were determined to a maximum resolution ranging between 1.55 and 2.05 å (1 å = 0.1 nm) and their refinement converged to good R-factors. This indicates that the structures can be used for a reliable analysis of the different interactions. Inspection of the electron density maps of the soaked structures revealed clear density for several sugar units. For the soaking experiments with xylotriose and xylotetraose respectively, three and four xylose units could be built in the electron density and the bound ligand atoms refined well with average thermal factors


Structure of Bacillus subtilis GH family 43 arabinoxylan arabinofuranohydrolase
Table 1 Data collection and refinement statistics

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Values in parentheses are for the highest resolution shell. AXH Additive Data collection ° Wavelength (A) Source ° Resolution range (A) Reflections Observed Unique Completeness (%) Mean I / (I ) Multiplicity R sym (%)* Crystal Space group Unit-cell parameters 30 % (w/v) Sucrose 0.9785 SLS X10SA 30­2.0 (2.11­2.0) 156621 35773 (4966) 96.4 (93.2) 14.1 (3.9) 4.4 (4.2) 10.2 (33.3) AXH xylotriose 1.0 M Lithium chloride 1.0788 DESY BW7a 50­1.55 (1.58­1.55) 258631 76939 (3868) 98.1 (99.9) 14.0 (3.9) 3.4 (3.1) 8.3 (32.2) 1.0322 DESY BW7a 50­2.05 (2.09­2.05) 150581 33537 (1596) 97.0 (93.9) 22.4 (8.0) 4.5 (4.2) 4.6 (13.7) AXH xylotetraose AXH AXOS-4-0.5 0.1 M Betaine hydrochloride 0.9785 SLS X10SA 30­1.8 (1.9­1.8) 390182 50933 (7349) 99.9 (99.8) 19.9 (6.1) 7.7 (7.5) 8.1 (27.9) AXH cellotetraose 30 % Sucrose 1.0011 SLS X10SA 30­1.8 (1.9­1.8) 306957 45414 (6055) 92.0 (85.4) 19.0 (5.4) 6.8 (6.6) 8.6 (29.2)

P a b c

21 2 1 2 1 ° = 67.6 A ° = 74.4 A ° = 107.0 A

P a b c

21 21 21 ° = 68.7 A ° = 73.7 A ° = 106.5 A

P a b c

21 21 21 ° = 67.5 A ° = 72.4 A ° = 106.9 A

P a b c

21 21 21 ° = 67.8 A ° = 74.2 A ° = 107.3 A

P a b c

21 21 21 ° = 68.0 A ° = 73.1 A ° = 105.7 A

Refinement R work (%) R free (%) Root mean square deviations ° Bond lengths (A) Bond angles ( ) Number of atoms Protein Solvent Ligand ° Average B -factor (A2 ) Main chain Side chain Solvent Ligand PDB entry

16.8 21.3 0.016 1.50 3671 311

16.4 19.0 0.010 1.22 3674 579 28 13.9 14.5 29.1 16.7 3C7F

15.1 17.4 0.012 1.50 3675 446 37 12.5 12.5 24.0 18.1 3C7G

15.5 19.0 0.015 1.44 3682 407 28 14.1 14.1 24.1 52.3 3C7H

15.1 17.5 0.012 1.50 3661 440 45 14.9 15.4 32.0 60.0 3C7O

16.1 16.0 24.4 3C7E

*R sym = |I (I )|/ I . R = ||F o |-|c |/ |F c |. The R free was calculated with 5 % of the data excluded from structure refinement.

comparable with the values for all protein atoms. Also for the crystals soaked in cellotetraose and AXOS-4-0.5, there was clear density for four glucose and four xylose units respectively, but no interpretable density for arabinose units could be observed for the structure soaked in AXOS-4-0.5. For these structures, the thermal factors of the sugars are slightly higher compared with those for all protein atoms (Table 1). Each asymmetric unit contains one BsAXH-m2,3 monomer, which is organized into two domains: the N-terminal catalytic domain, which is a five-bladed -propeller fold common to all GH family 43 members, and a C-terminal -sandwich domain, the CBM, analogous to CBM family 6 members (Figure 1). Superposition of the different structures revealed no large structural changes in the overall structure on ligand binding. Unlike the -xylosidase from G. stearothermophilus [14] and the bifunctional -xylosidase/ -L-arabinofuranosidase from S. ruminantium [17], BsAXH-m2,3 does not crystallize as a homotetramer, but as a single entity. This monomeric state of BsAXH-m2,3 is in contrast with the quaternary structure of other two-domain GH family 43 members for which the structure is known. The latter all occur as homotetramers, which are composed of two dimers turned at 90 against each other. The monomers forming the dimer are aligned antiparallel to one another such that the catalytic domain of one monomer interacts

with the CBM of the neighbouring domain. This arrangement results in a quaternary structure exhibiting 222 symmetry.

The catalytic domain

The catalytic domain forms a five-bladed -propeller, with the five -sheets (marked I­V) radially arranged around a central water-filled tunnel like the blades of a propeller. Each -sheet is built up of four antiparallel -strands connected by hairpin turns, with the first strand being the innermost. All five innermost -strands start at the same site, i.e. the entrance site, where the active site is located, and run almost parallel to one another. -Strands 2 and 3 of the five -blades are connected by large loops lining the active site (Figure 1b). Together with the loop regions connecting -strand 4 of one sheet to strand 1 of the next sheet, these loop regions probably have a role in determining the substrate specificity of BsAXH-m2,3. Unlike most propeller structures, BsAXH-m2,3 does not show the classical molecular velcro, which closes the propeller by incorporating the N-terminus and C-terminus in one blade of the propeller. However, some hydrogen bonds exist between the N-terminus and strand 4 of propeller blade V. These bonds probably provide stabilization to the fold. This non-velcroed propeller
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Figure 1

Overall structure of Bs AXH-m2,3

The catalytic domain is shown in blue, while the CBM is shown in green. The three catalytic residues are shown in ball and stick representation. (a) Top view showing the numbering of the five -blades (I­V). (b) Side view showing the position of the active site surrounded by the long loops connecting -strands 2 and 3 of each -blade.

has also been observed in the structure of other known GH family 43 members [15].
The active site and substrate binding

For members of GH family 43, three residues are essential for catalytic activity. For BsAXH-m2,3, these residues could be identified by superposing the structure of BsAXH-m2,3 with the structure of the -xylosidase from G. stearothermophilus [14]. They are: Asp-24, which is the general base; Glu-225, which is the general acid; and Asp-163, which is believed to play a role in the pK a modulation of the general acid and correct orientation of both the general acid and substrate. They are located on the innermost strands of -blades I, IV and III respectively and point to the centre of the tunnel entrance site. Seen from the surface, they are situated in a small pocket, in which a single arabinose substituent would fit well, along an open cleft, which allows binding of the xylan backbone. This arrangement of the active site is very suitable for the hydrolysis of arabinose substituents from mono-substituted xylose units and nicely illustrates why BsAXH-m2,3 does not hydrolyse arabinose from disubstituted xylose units. The pocket is too small to allow binding of two arabinoses. Since the crystals are grown from active BsAXH-m2,3, soaking with its substrate, AXOSs, should result in hydrolysis of the
c The Authors Journal compilation c 2009 Biochemical Society

arabinose substituents, and hence only the bound xylan backbone would probably be observed. Therefore soaking experiments were performed not only with AXOSs but also with short unsubstituted xylan chains to gain insight into the binding of the xylan backbone to BsAXH-m2,3. Three-dimensional complexes were obtained with xylotriose, xylotetraose, AXOS-4-0.5 and cellotetraose. The latter soaking experiment was performed to gain insight into the binding capacity of the CBM to cellulose, since some CBM family 6 members are found to bind cellulose [19]. Surprisingly, cellotetraose was bound to the active site. To determine the direction of the sugar backbone, the difference electron density map was contoured at a high level to observe primarily the positions of the oxygen atoms. Interestingly, for the complex structures, a glycerol molecule (originating from the cryo solution) is located at the probable position of the target arabinose and hence gives us a hint about the interactions with the arabinose unit in this subsite. The different complex structures reveal several residues responsible for binding interactions with the xylan backbone. Since BsAXH-m2,3 hydrolyses substituents of the xylan backbone, the subsite numbering proposed by Davies et al. [36] cannot be used to number the different binding subsites for the xylose units of the xylan backbone. So, for convenience, the binding subsites observed here are numbered I­IV starting from the xylose unit at the reducing end of the xylan backbone, with the III subsite being equal to the ­1 subsite in the numbering proposed by Davies et al. (Figure 2a). An arabinose substituent would be situated at the +1 subsite. Superposition of the different complex structures shows no difference in the position of the bound sugars and glycerol molecules (Figure 2b). When superposing the unbound structure with the complex structure, the side chain of Asn-288 turns by 90, away from the sugar. Apart from the xylose unit in the III subsite (from where the substituent is removed), only a few hydrogen-bonding interactions are observed between BsAXHm2,3 and the xylan backbone, of which all the xylose units are in the chair conformation (Figure 2). Two pronounced hydrophobic stacking interactions are observed with Phe-244 and Trp-160 and the xylose units in the II and IV subsites respectively, while the xylose unit in subsite I makes several hydrogen bonds with Gly-286. In the III subsite the OH2 of the xylose unit makes one hydrogen bond with Asn-288 (3.3 å) and two relatively strong hydrogen bonds with the general acid, Glu-225 (2.6 and 3.0 å), while the OH3 makes one hydrogen bond with Glu-225 (3.6 å). The extra carbonyl group of the glucose units from cellotetraose does not seem to make any additional interaction with BsAXH-m2,3 in comparison with xylotetraose. The binding of cellotetraose to the active site is probably an artefact based on the structural relationship between the xylopyranose and glucopyranose ring, since this binding has no physiological meaning, i.e. arabinocellulose does not occur in Nature. In the II and IV subsites, binding is solely based on hydrophobic stacking interactions. Since, in contrast with an extensive hydrogen-bonding network, hydrophobic stacking interactions allow more positional freedom, these interactions may account for the observation that either substituents at the O-2 or O-3 hydroxyl position of the xylose can be hydrolysed. This property of BsAXH-m2,3 needs some positional flexibility to allow correct positioning of the glycosidic bond with regard to the catalytic triad. Further studies involving soaking experiments with inactive BsAXH-m2,3 and specific AXOSs only substituted at their O-2 or O-3 hydroxyl position are required to confirm the above hypothesis. Since the hydroxy groups from glycerol usually take similar positions to the hydroxy groups from sugars, the interactions between the glycerol in the catalytic pocket and BsAXH-m2,3 give


Structure of Bacillus subtilis GH family 43 arabinoxylan arabinofuranohydrolase

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Figure 2

Detailed view of the active site of Bs AXH-m2,3

(a) The interactions between xylotetraose (white) and AXH-m2,3 are shown with hydrogen bonds represented as red dotted lines. The glycerol molecule occupying the catalytic pocket is shown in grey. The catalytic residues are in orange. (b) Superposition of the substrate molecules in the active site. The structure soaked in xylotriose is shown in blue, the one soaked in xylotetraose in pink and that with AXOS-4-0.5 in green, and the structure soaked in cellotetraose is shown in grey.

us an idea of the interactions with the arabinose unit in the catalytic pocket. Hydrogen bonds are formed with the three catalytic residues (Glu-225, Asp-163 and Asp-24) and Arg-321 (Table 2). In analogy with other GH family 43 members, Trp-101, which is part of the invariant Trp-Ala-Pro element, probably makes a hydrophobic stacking interaction with the arabinose unit [14].

The CBM

In analogy with other CBM family 6 members, the CBM of BsAXH-m2,3 forms a -sandwich fold consisting of five antiparallel -strands on one face and four antiparallel -strands on the other face (Figure 3). During refinement, the electron-density maps revealed residual density for metal ions at two positions

(Figure 3). The first metal ion could be modelled as a calcium ion and is hepta-co-ordinated by the side chains of Glu-359, Glu-361, Asn-383 and Asp-480. The co-ordination is completed by the backbone carbonyl oxygen atoms of Gln-384 and Asp480. The position and nature of this ion are identical with those found in other known CBM family 6 members and probably make it a structural ion [14,19,27,37,38]. It is likely to be already incorporated during protein folding, since no calcium ions were used during the purification and crystallization of BsAXH-m2,3, supporting the idea of it being a structural ion. The co-ordination of the second ion, modelled as a sodium, involves the side chains of Gln-390 and Asp-393. This co-ordination is completed by the main-chain carbonyl oxygen atoms of Arg-368 and Ser-388 and two water molecules. This metal ion corresponds to the sodium ion observed in the CBM family 6 of B. halodurans
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Table 2
Subsite I subsite

E. Vandermarliere and others
Interactions between Bs AXH-m2,3 and xylotetraose
Substrate atom O-2 O-2 O-3 O-2 O-2 O-2 O-3 O-1 O-1 O-1 O-3 O-3 O-3 O-3 Protein atom Gly-286 N Gly-286 CO Gly-286 CO Phe-244 Asn-288 N 2 Glu-225 O2 Glu-225 O1 Glu-225 O1 Trp-160 Glu-225 O1 Asp-163 O 2 Asp-163 O 1 Asp 24 O 1 Asp 24 O 2 Arg-321 NH2 Arg-321 NH1 Trp-101 ° Distance (A) 3.2 3.5 3.6 Hydrophobic stacking 3.3 2.6 3.0 3.6 Hydrophobic stacking 3.4 3.3 2.5 3.5 2.7 2.9 2.8 Hydrophobic stacking

II subsite III subsite (­1 subsite)

IV subsite +1 subsite (glycerol)

laminarinase and to the sodium ion observed in the CBM family 6of Cellvibrio mixtus endoglucanase 5A [27,38]. In contrast with the calcium ion, sodium might have been incorporated during the crystallization of BsAXH-m2,3 since the precipitant and cryo solutions contain sodium ions. Soaking experiments showed that neither cellotetraose nor xylo-oligosaccharides were bound to the CBM. This can tentatively be explained as follows. CBMs of family 6 contain two clefts that play a role in sugar binding (Figure 4a). Cleft A is found in the loop region connecting the inner and outer sheets of the -sandwich and resembles the sugar-binding sites of lectins [37], whereas cleft B is located on the concave surface of one -sheet and has a similar location to the binding

sites of CBMs from several other families [38]. When comparing cleft A of BsAXH-m2,3 with several other CBMs of family 6, BsAXH-m2,3 is clearly lacking the two aromatic residues that play a key role in ligand binding [39]. The amino acids at the equivalent position are Ser-388 and Ala-445 (Figure 4b). Cleft B is lined with two aromatic residues, Trp-394 and Phe465, which is in agreement with other CBMs belonging to family 6. However, in analogy with the CBM family 6 from Clostridium thermocellum xylanase 11A [19], the CBM family 6 from B. halodurans laminarinase [27] and the CBM family 6 from Clostridium stercorarium xylanase [37], a loop region covers this cleft, making it inaccessible for sugar (Figure 4c). These findings may explain the absence of binding of xylo-oligosaccharides and cellotetraose to the CBM of BsAXH-m2,3, since both binding clefts are structurally incapable of binding sugar chains. This CBM probably lost its carbohydrate-binding function during evolution. The loss of carbohydrate-binding capacity is also seen for CBMs of family 48. This family of CBMs is related to the starch-binding domains belonging to CBM families 20 and 21. Members of the latter families have binding sites that are lined with aromatic residues [40]. In analogy with cleft A of the CBM of BsAXH-m2,3, the carbohydrate-binding capacity of the CBM48 of glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus is also lost due to mutations of the aromatic residues that are responsible for carbohydrate binding [41].

Comparison of Bs AXH-m2,3 with other GH family 43 structures

The availability of the structure of this AXH-m2,3 and of other GH family 43 enzymes, in complex with their substrates, provides insight into how these structurally related enzymes are finetuned towards different substrate specificities. Indeed, despite their similar structures, reaction mechanisms and catalytic residue

Figure 3

Overall structure of the CBM and its bound metal ions

(a) Overall view of the CBM. The sodium ion is shown in purple, the calcium ion in orange. (b) Detailed view of the interactions between Bs AXH-m2,3 and the calcium ion. (c) Detailed view of the interactions between Bs AXH-m2,3 and the sodium ion. The water molecules involved in co-ordinating the sodium ion are shown as red balls.
c The Authors Journal compilation c 2009 Biochemical Society


Structure of Bacillus subtilis GH family 43 arabinoxylan arabinofuranohydrolase

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Figure 4 Structural differences at the binding clefts of CBM family 6 members
(a) Cartoon representation of the CBM of Bs AXH-m2,3 showing the position of both binding clefts A and B. (b) Detail of binding cleft A. The two aromatic residues with a key role in sugar binding are shown in ball and stick representation. The CBM6 of Bs AXH-m2,3 is shown in green, the CBM6 of Cl. thermocellum xylanase 11A (PDB entry 1gmm) [19] is shown in purple, the CBM6 of Ce. mixtus endoglucanase 5A (PDB entry 1uyz) [38] is shown in blue, the CBM6 of Bacillus haludorans laminarinase (PDB entry 1w9t) [27] is shown in orange and the CBM6 of C. stercorarium xylanase (PDB entry 1nae) [37] is shown in grey. (c) Detail of binding cleft B. The two aromatic residues that are important for sugar binding are shown in ball and stick representation. The CBM6 of Bs AXH-m2,3 is shown in green and the CBM6 of Cl. thermocellum xylanase 11A (PDB entry 1gmm) [19] is shown in purple.

Figure 5 Structural differences at the active site of several GH family 43 members
(a) Detail of the superposition of the active site of Bs AXH-m2,3 (blue), -L-arabinanase from Ce. japonicus (purple) and -xylosidase from G. stearothermophilus (green) in complex with their substrate, showing the perpendicular orientation of the substrate. (b) Cartoon representation of the superposition of Bs AXH-m2,3 (blue) in complex with xylotetraose (ba